Ricin Agglutinin Research Articles

A Comprehensive Review on Euphorbiaceae lectins: Structural and Biological Perspectives.

Euphorbiaceae, also known as the spurge family, is a large group of flowering plants. Despite being tropical natives, they are now widespread. Due to its medicinal and commercial importance, this family of plants attracted a lot of attention in the scientific community. The distinctive characteristic of the family is production of milky latex, which is a rich source of several lectins, the proteins that bind carbohydrates. Although their function is unclear, they are believed to defend plants against damaging phytopathogenic microorganisms, insects, and predatory animals. Additionally, they serve as crucial metabolic regulators under a variety of stressors. Detection, separation, purification, and characterization of lectins from the Euphorbiaceae family - mostly from the latex of plants - began over 40 years ago. This effort produced over 35 original research papers that were published. However, no systematic review that compiles these published data has been presented yet. This review summarizes and describes several procedures and protocols employed for extraction and purification of lectins belonging to this family. Physicochemical properties and biological activities of the lectins, along with their medicinal and pharmacological properties, have also been analyzed. Additionally, using examples of ricin and ricin agglutinin, we have structurally analyzed characteristics of the lectin known as Ribosome Inactivating Protein Type II (RIP-Type II) that belongs to this family. We anticipate that this review article will offer a useful compendium of information on this important family of lectins, show the scientists involved in lectin research the gaps in our knowledge, and offer insights for future research.

Assembly of Glycochips with Mammalian GSLs Mimetics toward the On-site Detection of Biological Toxins.

According to our previously proposed scheme, each of three kinds of glycosphingolipid (GSL) derivatives, that is, lactosyl ceramide [Lac–Cer (1)] and gangliosides [GM1-Cer (2) and GT1b-Cer (3)], was installed onto the glass surface modified with Au nanoparticles. In the present study, we tried to apply microwave irradiation to promote their installing reactions. Otherwise, this procedure takes a lot of time as long as a conventional self-assembled monolayer (SAM) technique is applied. Using an advanced microwave reactor capable of adjusting ambient temperatures within a desired range, various GSL glycochips were prepared from the derivatives (1)–(3) under different microwave irradiation conditions. The overall assembling process was programed with an IC controller to finish in 1 h, and the derived GSL glycochips were evaluated in the analysis of three kinds of biological toxins [a Ricinus agglutinin (RCA120), botulinum toxin (BTX), and cholera toxin (CTX)] using a localized surface plasmon resonance (LSPR) biosensor. In the LSPR analysis, most of the irradiated GSL chips showed an enhanced response to the targeting toxin when they were irradiated under optimal temperature conditions. Lac–Cer chips showed the highest response to RCA120 (an agglutinin with β-D-Gal specificity) when the microwave irradiation was conducted at 30–35 °C. Compared to our former Lac–Cer glycochips with the conventional SAM condition, their response was enhanced by 3.6 times. Analogously, GT1b chips gained an approximately 4.1 times enhancement in their response to botulinum type C toxin (BTX/C) when the irradiation was conducted around at 45–60 °C. In the LSPR evaluation of the GM1-Cer glycochips using CTX, an optimal condition also appeared at around 30–35 °C. On the other hand, the microwave irradiation did not lead to a notable increase compared to the former GM1-Cer chips derived with the SAM technique. Judging from these experimental results, the microwave irradiation effectively promotes the installing process for all the three kinds of the GSL derivatives, while the optimal thermal condition becomes different from each other. Many bacterial and botanic proteinous toxins are composed of such carbohydrate binding domains or subunits that can discriminate both the key epitope structure and the dimension of glycoconjugates on the host cell surface. It is assumed that the optimal irradiation and thermal conditions are required to array these semi-synthetic GSL derivatives on the Au nanoparticles in a proper density and geometry for tight adhesion with each of the biological toxins.

Protein Sensing Device with Multi-Recognition Ability Composed of Self-Organized Glycopeptide Bundle.

We designed and synthesized amphiphilic glycopeptides with glucose or galactose at the C-terminals. We observed the protein-induced structural changes of the amphiphilic glycopeptide assembly in the lipid bilayer membrane using transmission electron microscopy (TEM) and Fourier transform infrared reflection-absorption spectra (FTIR-RAS) measurements. The glycopeptides re-arranged to form a bundle that acted as an ion channel due to the interaction among the target protein and the terminal sugar groups of the glycopeptides. The bundle in the lipid bilayer membrane was fixed on a gold-deposited quartz crystal microbalance (QCM) electrode by the membrane fusion method. The protein-induced re-arrangement of the terminal sugar groups formed a binding site that acted as a receptor, and the re-binding of the target protein to the binding site induced the closing of the channel. We monitored the detection of target proteins by the changes of the electrochemical properties of the membrane. The response current of the membrane induced by the target protein recognition was expressed by an equivalent circuit consisting of resistors and capacitors when a triangular voltage was applied. We used peanut lectin (PNA) and concanavalin A (ConA) as target proteins. The sensing membrane induced by PNA shows the specific response to PNA, and the ConA-induced membrane responded selectively to ConA. Furthermore, PNA-induced sensing membranes showed relatively low recognition ability for lectin from Ricinus Agglutinin (RCA120) and mushroom lectin (ABA), which have galactose binding sites. The protein-induced self-organization formed the spatial arrangement of the sugar chains specific to the binding site of the target protein. These findings demonstrate the possibility of fabricating a sensing device with multi-recognition ability that can recognize proteins even if the structure is unknown, by the protein-induced self-organization process.

Detection and Quantification of Ricin in Beverages Using Isotope Dilution Tandem Mass Spectrometry

The toxic plant protein ricin has gained notoriety due to wide availability and potential use as a bioterrorism agent, with particular concern for food supply contamination. We have developed a sensitive and selective mass spectrometry-based method to detect ricin in tap water, 2% milk, apple juice, and orange juice. Ricin added to beverage matrices was extracted using antibody-bound magnetic beads and digested with trypsin. Absolute quantification was performed using isotope dilution mass spectrometry with a linear ion trap operating in product-ion-monitoring mode. The method allows for identification of ricin A chain and B chain and for distinction of ricin from ricin agglutinin within a single analytical run. Ricin-bound beads were also tested for deadenylase activity by incubation with a synthetic ssDNA oligomer. Depurination of the substrate by ricin was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS). This method was used successfully to extract ricin from each beverage matrix. The activity of recovered ricin was assessed, and quantification was achieved, with a limit of detection of 10 fmol/mL (0.64 ng/mL).

Ricin A Chain from Ricinus sanguineus: DNA Sequence, Structure and Toxicity

The gene coding for the ricin A-chain from Ricinus sanguineus (RsTA) was isolated and sequenced (GB: DQ661048). Comparison of RsTA with the ricin A-chain from Ricinus communis (RTA) revealed the presence of five differences in the gene sequence. At the protein level only two differences were noticed, the replacement of Asn136 by Ser (N136S) and Ile173 by Val (I174 V). From the RsTA structure model (PMDB: PM0074652), the N136S mutation was predicted to have no effect on RsTA structure. The I174V mutation is believed to have no effect on the RsTA structure or on toxicity since this replacement was found in the Ricinus agglutinin's A-chain and the latter has a comparable toxicity to RTA. The Ser221 of the putative lipase active site was found. Toxicity experiments showed that RsTA and RTA have similar toxicities. This finding proves that the N136S and I174 V mutations have no effect on RsTA toxicity.

Purification and anti-cancer activity of ricin

To extract and purify ricin from castor beans and to evaluate its anti-cancer activity. Ricin was purified from castor beans according the modified method of Nicolson and Blaustin. The lectins were extracted in 0.01 mol/L phosphate buffered saline and isolated in the 40% to 80% fraction of ammonium sulfate precipitation. The dialyzed fractionated preparation was applied with a Sepharose 4B column. The lectins were eluted with a linear lactose gradient (0.01 mol/L approximately 0.5 mol/L). Ricin was separated from the ricinus agglutinin by gel filtration on a Sephadex G-100. MTT was applied to analyze the cytotoxicity with different dosage of ricin in different cancer cell lines. There was no difference between the killing effect of normal cells and that of colon cancer cells by using the high dosage of ricin (5 x 10(-8) mol/L approximately 5 x 10(-10) mol/L). However, the cytotoxicity was significant different in those cells with the low dosage of ricin (5 x 10(-11) mol/L approximately 5 x 10(-13) mol/L). Meanwhile ricin had the similar cytotoxicity to leukemia cell K562 and colon cancer cell SW480. Ricin is able to kill tumor cells selectively at low concentration, but the selectivity does not appear at high concentrations.

Ricin, ricin agglutinin, and the ricin binding subunit structural comparison by Raman spectroscopy

Raman spectroscopy is used to study conformation-sensitive vibrational bands of the plant toxins ricin and ricin agglutinin and the ricin binding subunit in aqueous solution. The analysis of the Raman data yields the conformational state of the protein molecules differing from that predicted by the X-ray data. The differences and similarities in the conformational state of ricin, ricin agglutinin, and ricin binding subunit are discussed.

A simplified method to evaluate the acute toxicity of ricin and ricinus agglutinin

A brief method is described for the assessment of lethal toxicity of ricin and ricinus agglutinin using a modified LD 50 assay. With this test, it is possible to obtain an LD 50 using only 10 experimental animals. The equation for calculating the confidence limit of LD 50 is derived in this article. The LD 50 values obtained with the method were compared with data reported in the literature using classical methods. The results indicate that the simplified method for evaluating the acute toxicity of ricin and ricinus agglutinin gives reliable results. For scientific, economic and ethical reason the method is suggested to replace the traditional LD 50 assay for plant toxins.

Conformational difference between ricin and ricin agglutinin in solution and crystal.

Conformational difference between ricin and ricin agglutinin in solution and crystal.

Rotavirus Infection of MA104 Cells Is Inhibited by Ricinus Lectin and Separately Expressed Single Binding Domains

Various lectins were tested for blocking rotavirus infection of MA104 cells and it was observed that galactose-specific lectins were the most inhibitory. Of these Ricinus agglutinin was able to inhibit infection (by human and animal strains) at concentrations as low as 10−9 M. In addition, in a virus overlay protein blot assay Ricinus agglutinin competed with simian rotavirus SA11 for binding to solubilized MA104 proteins. Amino acid sequence comparisons revealed similarity between the ricin toxin B subunit (which contains two separate carbohydrate-binding motifs: single binding domains (SBD) 1 and 2) and rotavirus spike protein VP4. A filamentous phage display system was used to independently express the two binding domains and while SBD1 inhibited infection of MA104 cells by CRW8, NCDV, and to a lesser extent Wa, SBD2 blocked only CRW8 and NCDV infection. Furthermore inhibition of CRW8 infection was a direct result of phage inhibiting virus attachment to cells. When amino acid 248 within SBD2 was mutated from the ricin toxin to the Ricinus agglutinin sequence this phage clone showed reduced binding to galactose and was no longer able to inhibit virus infection. Thus, rotavirus recognizes galactose as an important component of the receptor on MA104 cells.

Preliminary crystallographic characterization of ricin agglutinin.

The quaternary structure of ricin agglutinin (RCA) has been determined by x-ray crystallography. The refined structure of ricin proved to be a successful search model using the molecular replacement method of phase determination. RCA forms an elongated molecule of dimensions 120 A x 60 A x 40 A with two A chains at the center and a B chain at each end. The A chains are covalently associated via a disulfide bridge between Cys 156 of both chains. Additional contacts at residues 114-5 stabilize the dimer interface. The covalent association of RCAA chains was confirmed by gel filtration under reducing and nonreducing conditions.

Defective galactofuranose addition in lipophosphoglycan biosynthesis in a mutant of Leishmania donovani.

A mutant cell line of Leishmania donovani (R2D2), previously selected for resistance to the cytotoxic lectin ricin agglutinin, was found to be totally deficient in the synthesis and expression of lipophosphoglycan, a dominant surface virulence factor. The metabolic defect in R2D2 parasites responsible for its lipophosphoglycan (LPG-) phenotype was investigated in this study. Following metabolic labeling of R2D2 parasites with either [3H]galactose or [3H]mannose, the main glycosylphosphatidylinositide product that accumulated was Glc-PO4-Man-Man-GlcN-lyso-1-O-alkylphosphatidylinositol (PI). The metabolic defect was further defined using a cell-free glycosylation system. When membrane preparations from wild-type cells were incubated with UDP-[3H]galactose and unlabeled GDP-mannose in the absence of exogenous acceptors, radiolabeled lipophosphoglycan was synthesized. The addition of exogenous Man-Man-GlcN-PI or Galf-Man-Man-GN-PI stimulated lipophosphoglycan synthesis in vitro. In contrast, when membrane preparations from R2D2 cells were incubated with exogenous Man-Man-GlcN-PI as an acceptor or in the absence of exogenous acceptor, the truncated glycosylphosphatidylinositide Glc-PO4-Man-Man-GlcN-PI was the main radioactive product synthesized. However, when exogenous Galf-Man-Man-GN-PI was added to the R2D2 in vitro system, radioactive lipophosphoglycan was synthesized. Collectively, these results indicate that the mutant R2D2 cells are unable to complete the assembly of the glycan core of LPG because of a defect in the synthesis of the "activated" galactofuranosyl donor or the lack of a functional galactofuranosyltransferase.

Galactose-containing epitopes on the surface of IgG model immune complexes are accessible for specific binding with the high molecular weight ligand, ricinus agglutinin, in solution—light-scattering studies

Immunoglobulin G (IgG) molecules contain covalently linked carbohydrate chains with galactose residues in their branched “antennae”. The ability of galactose-containing epitopes on the surface of IgG model immune complexes (IC) to interact with a high mol. wt ligand in solution has been elucidated. Different types of IgG model IC with pre-detennined molecular mass were mixed with Ricinus Agglutinin (RC1), which is known to bind specifically to galactose-containing oligosaccharides. The relative light-scattering increases (Δ I) in the reaction mixture were measured as a function of time. The galactose-associated epitopes of the IgG model IC were accessible for binding with RC1. The rate of the interaction between IgG model IC and RC1 was dependent on the molecular mass of the complexes; the larger the model IC molecular mass, the faster the rate of interaction. The binding of RC1 to IgG model IC was highly specific because it was completely abolished in the presence of lactose. The galactose-containing epitopes of monomeric IgG were also able to interact with RC1 but the kinetics of the interaction was much slower. We suggest than an increase in the density of the epitopes on the surface of the model IC, by close attachment of the IgG molecules, mainly determines the ability of galactose-containing epitopes to be recognized by RC1. The data presented support the importance of IgG glycans in recognition events of IgG by biologically active molecules.

Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75% of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90% of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the t1/2 values for RCAI binding, internalization and efflux were 5, 18 and 13-14 min, respectively. By comparing efflux with and without galactose present, we found that 60% of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4 degrees C became localized within more caveolae within 2.5 min of warming to 37 degrees C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.

A ricin agglutinin-resistant clone of Leishmania donovani deficient in lipophosphoglycan

A variant cell line of Leishmania donovani (named R2D2) has been selected for resistance to the cytotoxic lectin ricin agglutinin and shown to be defective in the synthesis of its major glycoconjugate lipophosphoglycan. Compared to the parental line, R2D2 cells showed a marked resistance to the toxic effects of ricin and an increased sensitivity toward concanavalin A. The synthesis of lipophosphoglycan by R2D2 cells was judged to be < 1% relative to that of wildtype cells as determined by incorporation of radioactive mannose and galactose and by electrophoretic and chromatographic analyses. Although lacking lipophosphoglycan, R2D2 parasites were capable of infecting cultured U937 macrophages.

Identification and characterization of a monoclonal antibody recognizing a galactose-binding domain of the toxin ricin.

A monoclonal antibody raised against purified ricin B chain, 75/3B12, blocked ricin toxicity 30- to 100-fold in vitro. The 75/3B12 IgG and F(ab')2 blocked ricin binding to cell surface galactose-containing receptors. The 75/3B12 Fab bound ricin D with a Ka of 10(7) M-1, and this binding was blocked by asialofetuin, lactose, and N-acetylgalactosamine--molecules which interact with the ricin galactose-binding site--but not by fetuin, sucrose, or glucose. The 75/3B12 Fab contained no detectable carbohydrate and, according to several lines of evidence, did not bind ricin via Ig carbohydrate determinants. The monoclonal antibody appears to recognize a galactose-binding site on ricin D via the variable region of the antibody. The 75/3B12 Fab bound ricin E only 1/50 as well as ricin D and bound the Ricinus agglutinin only 1/80 as well as ricin D. The antibody specificity indicates that structural differences exist in the galactose-binding sites of the Ricinus communis lectins. Abrin and other lectins which bind galactose or N-acetylgalactosamine were not significantly bound by the monoclonal antibody. In vitro, the antibody blocked the nontarget toxicity of immunotoxins similarly to lactose. However, in vivo, unlike lactose, the 75/3B12 antibody protected mice from ricin toxicity.

Interaction between human IgD and ricinus agglutinin.

The results show that monoclonal IgD reacts specifically with ricinus agglutinin (molecular weight 120,000) (RcAI). The reaction between IgD and RcAI was inhibited by galactose and lactose but not by glucose, indicating that the interaction is mediated by galactose containing carbohydrate units located in or near the sigma-hinge region. Affinity chromatography on RcAI-Sepharose 4B was successfully used to isolate monoclonal IgD from the IgD myeloma plasma.

In situ localization of lectin-binding glycoconjugates in the matrix of growth-plate cartilage.

In the distal hypertrophic zone of growth-plate cartilage, the pericellular matrix surrounding individual chondrocytes and the territorial matrix uniting chondrocytes into columnar groups are invaded by metaphyseal endothelial cells prior to osteogenesis. In the present study, lectin-binding glycoconjugates were analyzed in these two matrix compartments of growth-plate cartilage from Yucatan swine. Nine lectin-fluorescein conjugates were tested by a postembedment method on 1-micron-thick, nondecalcified, Epon-embedded sections. Chondrocytes in all cellular zones were surrounded by a pericellular matrix which showed positive binding for peanut agglutinin (PNA), ricin agglutinin (RCA-I), and soybean agglutinin (SBA). Binding by these lectins was sensitive to digestion with hyaluronidase, chondroitinase, and trypsin. Pericellular glyconconjugtes that bind RCA-I and concanvalin A (CONA) after periodic acid oxidation, and which were sensitive to trypsin but not to chondroitinase or hyaluronidase, were present in the hypertrophic cell zone. Within the territorial matrix, binding of lectins specific for galactose, N-acetylgalactosamine, and fucose showed gradients of intensity which became maximal at the last transverse septum. Lectin-binding histochemistry more precisely differentiated the microheterogeneity of glycoconjugate distribution within these two matrix compartments than has been possible with other histochemical techniques. Lectin-binding affinity is a potentially useful technique by which to isolate cartilage matrix macromolecules unique to specific cellular zones of the growth plate.

Interaction between rabbit IgG immune complexes and Ricinus agglutinin

Ricinus communis agglutinin covalently bound to Sepharose 4B (RcA-Sepharose 4B) was able to selectively bind immune complexes consisting of antigen (bovine serum albumin) and rabbit IgG antibody which were rich in antibody (i.e., high molecular weight complexes of approximately 2000 000). No interaction was recorded between RcA-Sepharose 4B and immune complexes consisting of the antigen plus rabbit IgG antibody which were rich in antigen (i.e., complexes with a molecular weight of 300 000–500 000). These results indicate that binding to RcA is due to the increased density of galactose residues in the high molecular weight polymeric antibody component of the soluble immune complexes.

Studies on the antitumor lectins isolated from the seeds of Ricinus communis (castor bean)

Three toxic proteins and one agglutinin were purified from the seeds of Ricinus communis by a simple and fast method using Sepharose 4-B affinity chromatography followed by Sephadex G-100 gel filtration. The weakly adsorbed ricins A and B were retarded and eluted with the buffer from the affinity chromatographic column, while ricin C and ricinus agglutinin had to be eluted with 0.1 M galactose. The molecular weights of ricins A, B, and C were about 62,000 and that of ricinus agglutinin was 120,000, estimated by amino acid compositions and SDS gel electrophoresis. They all possessed two non-identical subunits: A and B chains linked by one disulfide bond. Their ld 50 values were 4, 28, 14 and 112 μg per kg body weight of mice for ricins A, B and C and ricinus agglutinin, respectively. The amino acid compositions of the three toxins and their A and B subunits were very similar, but not identical, while ricinus agglutinin showed a different composition. Ricin A is a newly isolated lectin which has a strong inhibitory effect on the growth of tumor cells. By using cell cultures, it was demonstrated that the tumor cells were more sensitive to lectin than non-transformed cells, and that this could be caused by the higher binding affinity of lectin to tumor cells than to non-transformed cells.